A variety of protocols in the prior art have been employed for production of monoclonal and polyclonal antibodies to glycoproteins, to glycolipids and to tumor-associated antigens. For example, production of monoclonal antibodies can utilize whole cells, which are a highly immunogenic vehicle. This method, however, is fraught with the problem of a low efficiency in producing antibodies of interest. In addition, purified, soluble antigens are poorly immunogenic in mice, in contrast to the same immunogens in other species such as rabbits and sheep.
Presentation of an antigen in an insoluble form may improve immunogenicity of soluble antigen preparations in mice. For example, alum precipitation of carcinoembryonic antigen in some cases enhances immunogenicity. However, adjuvants are often required to achieve desired levels of antibody production. Traditionally, adjuvants, such as Freund's, have been admixed with soluble antigen preparations for immunizing rabbits or goats. However, adjuvant is oftentimes ineffective in mice; even though it may produce detectable titers of antibody, the yield of antigen-specific hybridomas is low. The present invention discloses an insolubilized immunogen that is effective in producing antigen-specific hybridomas.
Soluble immunogen preparations described in the prior art also usually require a great deal of effort for isolation of sufficient quantities of purified antigen. Most protocols for immunizing with soluble antigens utilize 50-100 .mu.g of purified antigen. Preparing this dosage of antigen involves considerable effort in biochemical purification, because most cell surface or cytoplasmic antigens are produced only in small amounts by cells. An advantage of the present invention is that it provides a process for enrichment of a given antigen from a heterogeneous mixture, thus reducing the need for purification. The term "enrichment," as used herein, means an increased concentration of the antibody or antigen of interest.
Another advantage provided by this invention is the production of monoclonal antibodies to epitopes on the same antigen that ar distinct from the epitope identified by the antibody used in preparation of the immunogen. The need for antibodies to multiple epitopes of an antigen has been previously demonstrated. For example, it has been shown that more than one epitope can exist within a glycoprotein species, and that different combinations of epitopes can be found on different subpopulations of glycoprotein molecules. Thus, not all epitopes have to be on all the glycoprotein molecules and/or have the same distribution within a given tissue. More specifically, the inventors have found that the degree of antigenic heterogeneity in a human melanoma-associated antigen system is dependent upon the epitope recognized by the monoclonal antibody (Morgan et al., Mol. Immunol. 23:193-200, 1986). One monoclonal antibody (MAb), that recognized an epitope displayed on most of the melanoma glycoprotein molecules, had less antigenic heterogeneity than a MAb that recognized an epitope present only on a subpopulation of glycoprotein molecules. Utilizing monoclonal antibodies to carcinoembryonic antigen (CEA), the CEA preparation can be subdivided into populations of molecules bearing unique epitopes. This also has been demonstrated with monoclonal antibodies to Class II histocompatibility antigens (HLA-DR antigens). Thus, the generation of unique antibodies to specific epitopes on a given antigen is useful for the optimization of therapeutic applications of monoclonal antibodies involving delivery of radioisotopes, drugs, or toxins to a target tumor.
As with antigen purification, the prior art method of producing monoclonal antibodies to specific epitopes of a given antigen is laborious. The prior art methodology involves immunization with soluble purified antigen isolated by a series of steps: production of large quantities of a given monoclonal antibody, purification of that antibody, insolubilization of that antibody on an insoluble matrix (e.g., Sepharose.RTM., an agarose gel, optionally cross-linked, in bead-form), and typically utilization of at least one other chromatography procedure (e.g., lectin affinity chromatography, ion exchange chromatography, gel filtration or hydrophobic chromatography) in combination with monoclonal antibody affinity chromatography to purify the antigen from a heterogeneous antigen mixture. This method is expensive and time-consuming, and requires large numbers of cells as the source of the impure antigen. In addition, once isolated, the soluble antigen preparation is poorly immunogenic in mice. Thus, there is a need in the art for more efficient and less time-consuming methods of generating monoclonal antibodies to epitopes of antigens distinct from those recognized by existing monoclonal antibodies. Within the present invention, the source of antigen need not be purified, and yet immunogenicity is enhanced.
There is also a need in the art for a method of producing monoclonal antibodies to antigens for which polyclonal antisera exist. If these polyclonal antisera do exist, although in small amounts, in low titers, or in polyspecific form (containing many antibodies of different specificities), they may be used in the present invention for production of monoclonal antibodies. In many cases, the amounts of these antisera are limited, are insufficient for antibody affinity chromatography, and are thus useless for immunization procedures involving soluble purified antigen. An additional benefit of the immunization process of the present invention is the ability to generate monoclonal antibodies of different specificity compared to the polyclonal antiserum used in the immunization.
There is also a need in the art for a method for efficiently producing monoclonal anti-idiotypic antibodies, which recognize the antigen-combining site of a monoclonal antibody (MAb) of murine origin. It has been postulated and subsequently verified in certain animal models that anti-idiotypic antibodies can act to regulate the immune response in both a positive and a negative manner. In certain instances, anti-idiotypic antibodies can substitute for purified antigen in the production of vaccines, thus eliminating the need for biochemical purification of a given antigen.
As shown in certain animal models, anti-idiotypic antibodies utilized as vaccines may prove useful in adjuvant therapy of cancer. The field is handicapped by difficulties in producing monoclonal antibodies of murine origin to other murine monoclonal antibodies. Murine MAbs to human and rabbit antibodies have been successfully produced. However, most MAbs to tumor-associated antigens are of mouse origin. The combining site of these tumorspecific murine MAbs may be poorly immunogenic, presumably due to self-tolerance induced by common sequences within the Fc region. It appears that species distinction within the Fc region may result in enhanced immunogenicity of the antigen combining site (idiotope). In the present invention, the use of an immunosorbent enhances the generation of antibodies to the idiotope of a monoclonal antibody.
Another advantage of the present invention is that it provides an efficient technique for generating a battery of monoclonal antibodies that are directed against multiple epitopes of a given antigen. This methodology facilitates the production of monoclonal antibodies that recognize immunorecessive epitopes of an antigen. As a result, a variety of monoclonal antibodies may be bound to one target cell or antigen, thereby enhancing the delivery of a higher dose of conjugated toxin or radioisotope to a target site. Delivery of an increased concentration of immunoconjugates improves cytotoxicity or imaging properties, because multiple distinct epitopes of a target cell can be bound. In addition, the availability of a multiplicity of monoclonal antibodies will permit targeting of a variety of epitopes, some of which may be unique to a particular target cell. This unique binding capability may allow selection of monoclonal antibodies that exhibit little or no cross-reactivity with epitopes of normal cells, which in turn might permit administration of higher doses of immunoconjugates.